Transcript Gonzalez-Martinez SNP Genotyping

SNP Genotyping
Santiago C. González-Martínez
Center of Forest Research, INIA, PO Box 8111 28080 Madrid, Spain
[email protected]
Syvänen (2001)
Nature Genetics
DGGE and SSCP
(eco)Tilling
Single-base
extension
Fluorescence Polarization with
Terminator-Dye Incorporation (FP-TDI)
- a method for SNP detection
Fluorescence polarization
Chen et al. 1999
Genome Res.
FP-TDI Assay
What the data might look
like… if you are lucky
Gene 4CL
Usually, the data is not quite
as ‘perfect’…
Gene EREBPS
FP-TDI
ADVANTAGES
DISADVANTAGES
• No special primers
• Relatively simple procedure
• Results are immediate
•
Expensive
plate reader = $30, 000
kit = $10, 000 for 100, 000x
plates = $6 each
cost per reaction = 75c each
thermocycler maintenance
PCR (ie mostly Taq) costs
•
•
One SNP per reaction
Need to know sequence
Pyrosequencing
Microarrays
Syvänen (2001)
Nature Genetics
High-throughput methods
Syvänen 2005
Nature Genetics
Illumina- BeadStation500G-BeadLab Platform
How does it work?
Experimental Procedure
Example of a “nice” SNP
Examples of midrange SNPs
Above threshold:
GT = 0.7849
Cluster_sep =0.272
Below threshold:
GT = 0.7788
Cluster_sep =0.267
4CL-s5[c/t]
AT-s3[g/a]
Not resolved with FP
Better separation in Illumina
Illumina- BeadStation demo